1. Field of the Invention
The present invention relates to a method for detecting and analyzing a trace substance by utilizing the agglutination assay, in which an analyte reacts with a particle-labeled anti-analyte, such as an antibody, to cause the particle agglutination. Particularly, the present invention relates to a dry analysis method for determining an analyte, which comprises bringing a solation agent into contact with a medium of a non-fluid substance to increase the fluidity of the medium, thereby causing the agglutination of the particles bearing the anti-analyte in the medium. Also, the present invention relates to a dry analysis element which enables such analysis method.
2. Description of the Related Art
In recent years, it has come to be very important to quantitatively analyze a trace substance, particularly antibody or antigen, in a specimen promptly, conveniently and precisely in order to diagnose the condition of diseases or judge the effects of treatment. For this purpose, widely employed has been an immuno-serological test for assaying the existence of an antigen or antibody in the body fluid, in which the antibody or antigen is adsorbed and immobilized to insoluble carrier particles and the resulting particles are reacted with the antigen or antibody.
The latex particles agglutination immunoassay is performed routinely by mixing a suspension of antibody-coated latex particles (sensitized latex) with a specimen on a glass plate. The latex particles agglutinate, or fail to agglutinate, as a result of interacting with the analyte antigen in the specimen. The extent of the agglutination can be determined by visual inspection. This assay makes it possible to semi-quantitatively analyze the antigen in the specimen by diluting the specimen at various ratios similar to another qualitative assay.
In Japanese Patent Publication Nos. 11575/1983, 43138/1987 and 55013/1987, there is proposed a method, in which latex particles having an antibody bound thereto is reacted with the antigen in the sample and the amount of the agglutination of the latex particles is determined optically by nephelometry. According to the proposed method, an antigen or antibody has come to be analyzed quantitatively by an automatic analyzer.
In addition, Unexamined Japanese Patent Publication (KOKAI) Nos. 141665/1990 and 209879/1993 disclose a method wherein an antigenic substance is detected by measuring a change in the absorbance upon the agglutination of the colloidal gold-labeled antibodies.
The above-described immunoassays do not need B/F separation and in this point, they are useful. The latex reagent is, however, poor in storage stability, since it is in the liquid form. In the colloidal gold agglutination, the colloidal gold solution or dispersion is not suitable as a reagent because of poor storage stability. A colloidal gold-labeled reagent in the lyophilized form must be mixed with a dedicated solution upon measurement, which makes the operation cumbersome. This method is also accompanied with such a drawback as unsuitability for use in the measurement of a small amount of a sample.
A so-called dry analysis method is, on the other hand, superior in storage stability and convenient operation. The so-called wet system (or solution system) comprises dissolving a reagent to be used for the assay in an aqueous solvent, thereby preparing the corresponding reagent solution, adding this reagent solution to a sample to be analyzed and then measuring the color reaction product by a calorimeter, while the dry analysis method comprises spotting an aqueous sample directly to a dry analysis element, such as test piece, analytical slide or analytical tape, having a reagent composition incorporated therein in the dry form and effecting colorimetry of the color development or color change occurring in the element. The dry system is superior to the wet system using a reagent solution in convenient operation and speedy assay.
A method for causing agglutination in the layer of a dry analysis element, thereby directly detecting the existence of an agglutinate itself in the layer construction has not yet been proposed. It can adopt the agglutination in a gel state, like as the Ouchterlony technique, which involving immunoprecipitation through an agar gel. Ouchterlony test is one of immunodiffusion methods, in which an antigen and an antiserum diffuse in the agar gel from each of two holes made in the gel plate. The antigen and the antiserum meet each other to form a visible precipitation line. Long time duration is necessary for the test, since the diffusion lasts for some time.
In order to cause agglutination of the labeling carrier in a gel medium in short time, the gel which is to be a place for reaction is required to have fluidity sufficient for causing agglutination. If sufficient humidity is required for maintaining this fluidity, such an analysis element cannot be classified as an element stored in a dry condition (or semi-dry condition). Moreover, when the gel has high fluidity, it needs a special care for packaging or storage, which makes this method far from convenience.
The present invention has been accomplished in view of the aforementioned circumstances, and a first object of the present invention is to provide a dry analysis method for determining an analyte using an agglutination of the particles bearing an anti-analyte, by which a highly sensitive analysis is ensured while using a simple operation and a reagent can be stored with a higher stability in the dry state.
A second object of the present invention is to provide a dry analysis element which can detect agglutination caused by the reaction between an analyte and an anti-analyte labeled with labeling particle, thereby analyzing the analyte in a convenient and highly sensitive manner.
The first object of the present invention is attained by an agglutination assay method for quantitatively determination of an analyte in an aqueous liquid sample using particles bearing an anti-analyte, the anti-analyte being capable of specifically binding to the analyte so as to cause agglutination of the particles, comprising:
providing a mixture of said particles and a non-fluid substance which retains said particles while suppressing the diffusion of said particles;
contacting said mixture with a solating agent for increasing the fluidity of the non-fluid substance in said mixture;
contacting the sample with said mixture to cause the agglutination of the particles in said mixture; and
measuring the extent of the agglutination of the particles to determine the amount of the analyte in the sample.
In the invention, a non-fluid substance is used as a medium which is to be a place for the agglutination of the labeling carrier or particle bearing an anti-analyte. Upon analysis, the fluidity of the medium is enhanced by the solation agent. In other words, diffusion properties of the labeling particle is enhanced, and accordingly the diffusion and agglutination of the labeling particles is accelerated. By mixing the labeling carrier or particle with the non-fluid substance to form a medium, the medium is, upon storage, dry enough not to impair the stability of a reagent composition to be employed. While upon analysis, the medium is solated and liquefied by the solation agent and has acquired fluidity sufficient for causing agglutination of the labeling particles.
In a preferred embodiment, a solation agent is added to an aqueous liquid sample containing an analyte and then, a non-fluid medium containing a particle-labeled anti-analyte is brought into contact with the resulting mixture. In case where this medium is formed in a membrane or film form, the agglutinate formed in the medium can easily be detected by measuring an optical change of the transmitted or reflected light from outside of the film-like medium. The existence of the agglutinate and its amount may be detected as a turbidity change in the film-like medium or as a change in the color tone of the labeling particle due to agglutination.
The second object of the present invention is attained by a dry analysis element for quantitatively determining an analyte in an aqueous liquid sample by measuring the extent of agglutination of particles bearing an anti-analyte, the anti-analyte being capable of specific binding to the analyte to cause the agglutination of said particles, comprising:
a non-fluid medium layer composed of a non-fluid substance which retains said particles bearing the anti-analyte therein while suppressing the diffusion of said particles; and
a water permeable layer which is superimposed on said non-fluid medium layer and contains a solation agent being capable to increasing the fluidity of the non-fluid substance;
whereby, when the sample is applied to the water permeable layer, said solation agent transfers to the non-fluid medium layer from the water permeable layer together with the sample and increases the fluidity of said non-fluid substance to cause the agglutination of the particles in the non-fluid medium layer.
In the second aspect of the present invention, the non-fluid medium layer is used as a field where the agglutination takes placce. Upon analysis, the solating agent migrates from the upper water permeable layer to the lower non-fluid medium layer together with liquid sample applied to the element. The solating agent solates or liquefys the non-fluid substance of the non-fluid medium layer to enhance the diffusion of the particles and thereby the agglutination of the particles is accelerated. This constitution makes it possible to store the element under dry conditions sufficient for not damaging the stability of a reagent composition to be employed. Upon analysis, the non-fluid substance is solated and liqufied by the action of the solating agent, thereby maintaining fluidity sufficient for causing agglutination of the labeling particles.